ABSTRACT Staphylococcus aureus in an important bacterial pathogen that provokes a diverse range of infections, ranging from localized skin and soft tissue infections to invasive, life-threatening diseases, such as bacteremia, endocarditis, and necrotizing pneumonia. Acquisition of antibiotic resistance has made treatment of staphylococcal infections challenging. Development of an effective vaccine to prevent invasive diseases caused by S. aureus remains a healthcare priority, but success has been elusive as evidenced by four failed phase III clinical trials in humans. Recent studies have suggested that a vaccine that targets multiple bacterial antigens may prove to be the best strategy for producing broad and effective protection against S. aureus disease. The overall goal of this project is to develop a safe and effective S. aureus vaccine that stimulates a robust antibody and T cell response in the host. S. aureus has recently been shown to produce nano-sized, non-living extracellular vesicles (EVs) both in vitro and in vivo. These membrane-bound vesicles package multiple staphylococcal antigens, including those that are cytoplasmic, membrane-associated, and secreted in the parental strain, making it feasible to generate designer EVs as a multivalent vaccine platform. Moreover, EVs are enriched for lipoproteins that serve to enhance EV immunogenicity, and this may abrogate the need for administration with an adjuvant. A limitation of this vaccine approach is that S. aureus EVs also contain native toxins and immune evasion factors like staphylococcal protein A that can harm the host and dampen the antibody response. The hypothesis of the proposal is that genetically engineered S. aureus cells can be utilized to produce EVs that are safe, immunogenic, and elicit a protective immune response in the host. To address this, an accessory gene regulator and protein A double mutant (?agr?spa) of USA300 S. aureus strain JE2 was constructed. EVs prepared from the mutant strain were nontoxic, immunogenic, and elicited protective efficacy in a mouse sepsis model. Engineered EVs (eng-EVs) were generated that also package nontoxic alpha toxin and a leukocidin subunit. Mice immunized with the eng-EVs produced both binding and toxin neutralizing serum antibodies. The specific aims of this R21 application are to: 1) Characterize and further modify S. aureus extracellular vesicles to contain additional protective antigens. 2) Evaluate the immunogenicity and protective efficacy of second-generation engineered EVs in murine models of S. aureus infection. The humoral and T cell responses of immunized mice will be measured, and the animals will be challenged with diverse S. aureus strains in models of sepsis and surgical wound infection. At the conclusion of these studies, creation of a novel genetically engineered EV vaccine platform will be accomplished, and these eng-EVs will be tested for protective efficacy as a multivalent vaccine for the prevention of S. aureus infections. These studies may also allow the identification of previously unknown S. aureus protective antigens.